Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure

Abstract

C-terminal brain (B-type) natriuretic peptide (BNP)-32 is a widely used clinical biomarker for the diagnosis, prognosis, and treatment of heart failure (HF). The 32-aa peptide is synthesized primarily in the atrial and ventricular myocardium and constitutes the mature biologically active form of immature BNP (pro-BNP). There has been mounting evidence that suggests BNP circulates in different structural forms that impact HF diagnosis and in vivo activity. Herein, we have developed and used an immunoaffinity purification assay to isolate endogenous BNP-32 from New York Heart Association class IV patient plasma for subsequent analysis by nano-liquid chromatography (LC) electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS. We have introduced stable isotope-labeled BNP-32 to the assayed plasma to enable quantification of endogenous levels of BNP-32. Unlike the chemically nonspecific point-of-care tests (POCTs) and RIAs used worldwide to quantify BNP-32 from plasma, FT-ICR-MS (unprecedented mass measurement accuracy) coupled with LC (retention time) affords extraordinary molecular specificity, and when combined with the use of internal standards is able to confidently identify and quantify BNP-32. The significance of this work is despite exceedingly high circulating levels of BNP-32 in the New York Heart Association class IV patients as determined by POCTs (>290 fmol/ml) nano-LC-electrospray ionization-FT-ICR-MS data did not reveal any endogenous BNP-32. These results provide molecularly specific evidence for the absence of circulating BNP-32 in advanced-stage HF patients and suggest the existence of altered forms of BNP that are contributing to the POCT values.

Fig. 1.

Experimental approach for determining the standard curve for BNP-32. (a) Chemical structure of stable isotope-labeled BNP-32 (BNP-32^*). (b) Standard curve for BNP-32 using data derived from nano-LC-ESI-FT-ICR MS data. A representative FT-ICR mass spectrum of the 5+ charge state from a 1:1 mixture (500 fmol each) of exogenous BNP-32 and BNP-32^* is shown as an example. The ion abundances used for determining the standard curve were defined by using the A + 2 isotope from both species (denoted by ○). RSD, relative standard deviation.

Fig. 2.

Experimental approach for developing and optimizing the dual-antibody immunoaffinity purification assay for nano-LC-ESI-FT-ICR MS analysis. The general protocol was as follows: (i) 500 fmol of exogenous BNP-32 was added to 1 ml of healthy patient plasma (<5 fmol/ml endogenous BNP-32) and passed through a C₈ solid-phase extraction (SPE) cartridge; (ii) plasma is eluted and then treated with the primary antibody (rabbit anti-BNP-32); (iii) after a period, the secondary antibody is added (goat anti-rabbit IgG) and incubated; (iv) the magnetic beads with the antigen-primary-secondary complex are separated, washed, and then released; (v) the dissociated complex is filtered and spiked with 100 fmol of BNP-32^*; and (vii) the resulting sample is reconstituted and analyzed by nano-LC-ESI-FT-ICR MS.

Fig. 3.

Heat map showing the percentage of BNP-32 recovery from plasma as a function of incubation time and primary antibody concentration. The FT-ICR mass spectrum of recovered exogenous BNP-32 ([BNP-32 + 5H⁺]⁵⁺) and the BNP-32^* ([BNP-32^* + 5H⁺]⁵⁺) internal standard under the optimal recovery conditions is shown to the left of the heat map.

Fig. 4.

Nano-LC-ESI-FT-ICR MS total ion chromatograms of 50 fmol BNP-32/500 fmol BNP-32^* in PBS, pH 7.4 (a) and 500 fmol of exogenous BNP-32 immunoaffinity-purified from plasma obtained from a healthy individual (b). The left y axis is the total ion current in arbitrary units (a.u.), and the right y axis is the percentage of B mobile phase (%B) over the course of the nano-LC gradient. FT-ICR mass spectra shown in b are representative of the high-molecular-weight species present in the immunoaffinity-purified sample. Mass-to-charge (m/z) values for the most abundant isotope and the approximate neutral masses are shown for each isotopic distribution. The total gradient time was 50 min (shown is 0-35 min containing the relevant elution window).

Fig. 5.

Sample collection, storage, and analysis protocol for NYHA class-IV patients (Table 1). (a) Patients present at the Mayo Clinic with HF and are identified by POCT to have exceedingly high levels of BNP-32 (>290 fmol/ml). (b) Patients return for follow-up visit, and multiple blood samples are drawn, processed into plasma, and frozen at -86°C. A 1-ml plasma sample is thawed, spiked with 500 fmol of the BNP-32^* standard, processed by using the dual-antibody immunoaffinity purification assay for BNP-32, and analyzed by nano-LC-ESI-FT-ICR MS. BNP-32 is measured by POCT in the second frozen sample after nano-LC-ESI-FT-ICR MS analysis to account for degradation.

Fig. 6.

Absolute quantitative nano-LC-ESI-FT-ICR mass spectral results from immunoaffinity-purified NYHA class-IV patient samples with 500 fmol of BNP-32^* internal standard. The left y axis is fmol of BNP-32/ml of plasma, and the right y axis is pg of BNP-32/ml of plasma. All three spectra show significant ion abundances for the BNP-32^* standard ([BNP-32^* + 5H⁺]⁵⁺) (500 fmol/ml) but no endogenous BNP-32 was detected by nano-LC-ESI-FT-ICR MS in any of the samples. The arrows represent the expected ion abundances for endogenous BNP-32 based on the POCT. Spectra are for patient 1 (a), patient 2 (b), and patient 4 (c). ^* in c denotes the isotopic distribution for a high-molecular-weight species that coeluted with BNP-32^*.